Technical support

analysis

You are working with HPLC for the separation and identification of analytical substances in laboratories. Challenge the selectivity of Kromasil for your most sensitive analysis.

development

You need to quickly purify substances for further studies in the development of pharmaceuticals. Kromasil will help you achieve your goals with ease and efficiency.

You are using preparative chromatography for high efficiency purification in a pilot or up to industrial-scale production. The robustness of Kromasil is available in ton quantities to meet your process demands.

Packing instruction for Kromasil in DAC columns

Always use clean frits, preferably new (outlet and inlet 3-5 µm porosity). If the frits are clogged, clean them with sodium hydroxide overnight, and wash carefully afterwards with water and alcohol. A frit can easily be clogged if an irregular silica is used.

Select the right slurry solvent. Below is a list of solvents that have been successfully used to generate efficient column beds.

Table 1 - Slurry solvent
Bare silica, 60 Å and 100 Å	C4/C8	C18	CN	DMB/TBB
Toluene/ 2-Propanol (50/50)	Acetone/ 2-Propanol (50/50)	THF/ 2-Propanol (50/50)	Anhydrous Ethanol	Toluene/ 2-Propanol (50/50)
Acetone/ 2-Propanol (50/50)	Anhydrous Ethanol	Toluene/ 2-Propanol (50/50)		Chloroform/ 2-Propanol (50/50)
		Chloroform/ 2-Propanol (50/50)

From the desired bed length and packing density given in the data sheet, calculate the amount of packing material needed for the column. For making up the slurry use at least 2 liters of slurry solvent per kg of packing. Calculate the amount of solvent by adding the total porosity and the excess for making up the slurry. Total porosity is the porosity of the particles and the interstitial porosity (space in between the particles), and is given in the table below. Make sure that there is some margin so that all slurry goes into the column.

Table 2 - Packing density
	Bare silica, 60 Å and 100 Å	C4	C8	C18	CN	DMB/TBB
	0.45	0.55	0.60	0.66	0.48	0.66

Table 3 - Total porosity
	Bare silica, 60 Å and 100 Å	C4	C8	C18	CN	DMB/TBB
	0.82	0.72	0.67	0.58	0.72	0.67

Make sure that the air tubing for the air supply to the hydraulic pump is large enough (see table 4)

Table 4 - Adequate air tubing dimension
	column size ID, mm	Air tubing ID, mm
	50	≥ 8
	150	≥ 15

Before starting the packing make sure that the solvent tubing is large enough. For 5 cm columns use at least 2 mm ID (1/8 inch, OD) outlet tubings during the packing procedure, and then change to smaller solvent tubings during chromatography (e.g. 1 mm ID for 5 cm columns). For larger columns scale the tubing accordingly to the diameter.

Plug the solvent inlet tubing during the packing procedure.

Pour the slurry into the column, and attach the lid. Start packing using the highest possible pressure (usually 70 or 100 bar). If the air supply is large enough the packing should be completed in about 30 seconds for a bed length of 25-30 cm.

For reversed phase chromatography do not use teflon membranes. These can cause problems due to poor wettability.

During chromatography decrease the piston pressure to 10-20 bars above the back pressure in the column.

For testing, preferably inject via a loop, and have the delivery system well optimized, using small dead volumes. For test substances see table 5.

Table 5 - test substances
	Reversed phase	Bare silica and Chiral
	Acetophenone, toluene, etc	Nitrobenzene, aromatic esters, etc

· Eka Chemicals, Separation Products · SE–445 80 Bohus, Sweden · Phone +46 31 58 70 00 · Fax +46 31 58 77 27 ·
· E-mail kromasil@akzonobel.com ·